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UFRJ – Instituto de Biofísica Carlos Chagas Filho, CCS – Bloco G, Rio de Janeiro, Brazil
Correspondence
Fernando Costa e Silva-Filho
fcsf{at}biof.ufrj.br
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The related pathology, usually referred to as bovine trichomoniasis, includes different clinical manifestations between bulls and cows (BonDurant, 1997). In bulls the parasite can be detected at the preputial cavity to the urethra, and also in deeper parts of the urogenital tract (Parsonson et al., 1974). Once infected, bulls may harbour T. foetus throughout their lives without exhibiting clinical symptoms. In contrast, among cows the effects of the disease range from asymptomatic to severe clinical manifestations including vaginitis, cervicitis, endometritis and pyometra, resulting in transient infertility or fetal loss. Such clinical characteristics indicate the ability of T. foetus to invade host tissues (Parsonson et al., 1976; López et al., 2000). Trichomonads possess a diversity of surface glycoconjugates including adhesins (Corbeil et al., 1989; Singh et al., 1999; Alderete & Garza, 1988) and extracellular matrix (ECM)-binding molecules (Silva-Filho et al., 1988), which play important roles during the interaction of the parasites with their hosts.
In mammals, the ECM is composed of a complex assortment of glycoproteins and proteoglycans, which not only serve as scaffolds to provide a structural framework for tissues but also regulate cell behaviour (Bissell & Barcellos-Hoff, 1987; Nelson & Bissel, 2006). Among the ECM components, laminin-1 (LMN-1) is a large (
850 kDa) glycoprotein involved in various biological phenomena (Ekblom et al., 2003). LMN-1 provides signalling cues mediating important cell functions, including cell adhesion (Arrighi & Hurd, 2002; Ghosh et al., 1999; Gordon et al., 1993; Silva-Filho et al., 2002) and invasiveness by micro-organisms (Bandyopadhyay et al., 2001; Li et al., 1995).
Biochemical studies focusing on the binding of eukaryotic cells to LMN-1 and the chemical nature of the related cell surface receptors have shown that the binding of some eukaryotic cells to LMN-1 is modulated by integrins (Mercurio, 1995; Hynes, 2002) and requires the participation of certain cationic species. The functionality of integrins is indeed revealed only in the presence of minimum amounts of each one of Mn2+, Ca2+, Mg2+, or both Ca2+ and Mg2+ (Plow et al., 2000). In addition, it has been found that the oligosaccharide moieties of LMN-1 (Fujiwara et al., 1988) and their lectin-like counterpart molecules residing on pathogen surfaces (Ferreira et al., 2006; Saarela et al., 1996) may mediate both recognition and binding of micro-organisms to LMN-1.
Since ECM binding by prokaryotic and eukaryotic pathogens may trigger pathogenesis and tissue invasion (Li et al., 1995; Hernandez-Ramirez et al., 2000; Meza, 2000), the aim of this study was to characterize the binding of T. foetus to immobilized LMN-1, including the molecular nature of the related receptor and the role played by parasite–LMN-1 binding in the interaction between T. foetus and cultured mammalian cells.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), was used throughout. The parasites were cultured in modified TYM medium (Diamond, 1957; Lockwood et al., 1984) supplemented with 10 % fetal calf serum at 37 °C. Parasite density and viability were estimated by counting in a haemacytometer chamber using erythrosin B as a viability marker (Philips, 1973). Only parasite suspensions exhibiting viability indexes higher than 90 % were used in the following experiments.
LMN-1 immobilization on glass surfaces.
Pre-cleaned glass slides were uncoated or coated with 5, 10, 20, 30 or 40 µg LMN-1 ml–1 (LMN from Engelbreth–Holm Swarm Tumour; Sigma) (Silva-Filho et al., 1988) made in Ca2+-, Mg2+- and Mn2+-containing 50 mM HEPES free acid, pH 6.6, for 2 h at 37 °C. Subsequently, the unbound LMN-1 was aspirated and discarded.
[3H]Thymidine labelling of T. foetus.
Parasites cultured at 37 °C for 24 h were collected by centrifugation, washed and ressuspended in TYM medium plus serum containing 30 µCi [3H]thymidine (Alderete et al., 1995; Rocha-Azevedo et al., 2005). After 24 h incubation the resulting radiolabelled parasites were collected by centrifugation (450 g), and washed three times with 0.01 M phosphate-buffered 0.145 M NaCl (PBS), pH 7.2, in order to remove the unassociated radioactive tracer.
Binding to immobilized LMN-1.
[3H]Thymidine-labelled parasites were suspended in 10 mM HEPES plus 125 mM sucrose containing 0.01 M CaCl2, MgCl2 and MnCl2, or without divalent cations, followed by an additional incubation with or without 5 mM EDTA. The parasites were allowed to interact with uncoated and LMN-1-coated slides for 1 h at 37 °C. In some assays sucrose was replaced by 125 mM β-lactose or galactose. Following interaction with LMN-1, the unbound parasites were aspirated and discarded; those which remained associated with the slides were washed twice and solubilized by incubation in a lysis solution (0.2 % SDS in 0.2 % NaOH). Radioactivity (c.p.m.) was measured by liquid scintillation. The resulting c.p.m. values were converted to the percentage of parasites attached to LMN-1 according to Rocha-Azevedo et al. (2005): % attachment=(c.p.m.experimental/c.p.m.positive control)x100.
Scanning electron microscopy.
Parasites were allowed to interact with glass coverslips either coated with 20 µg LMN-1 ml–1 or without LMN-1 for 60 min at 37 °C, and then carefully washed three times with PBS following fixation with 2.5 % glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2. The samples were then washed sequentially with PBS, post-fixed with 1 % osmium tetroxide in 0.1 M sodium cacodylate buffer (pH 7.2), dehydrated in a graded series of ethanol, critical-point dried by using CO2, and sputter-coated with gold. The samples were examined in a JEOL JSM-5310 scanning electron microscope. The morphological types found among the parasites were scored and counted as follows: tear-shaped or slightly round (not amoeboid) and spread (amoeboid), in 10 randomly chosen fields at 1000x magnification.
Trypsin and periodate treatments.
Untreated parasites and parasites treated with 3 mg trypsin ml–1 for 30 min at 37 °C, and parasites treated with 1 mM sodium periodate for 30 min at 4 °C (Crouch & Alderete, 1999), were washed with PBS, and their viability indices estimated as previously detailed.
Dot blot analyses.
Parasites were disrupted in lysis buffer (Galán et al., 1992) and the resultant cell extracts were then incubated with or without 10, 50 or 100 mg trypsin ml–1 for 15 min at 37 °C or with 5, 10, 20 or 50 mM sodium periodate for 30 min at 4 °C. The untreated and treated extracts were then blotted onto nitrocellulose membranes and incubated overnight with TBST blocking buffer (Tris-buffered saline with 0.1 % Tween, 20.5 % non-fat dry milk and 1 % BSA). Just after this, the membranes were sequentially loaded with 10 µg LMN-1 ml–1 for 2 h in Tris-buffered NaCl (TBS) containing 1 % BSA plus 0.01 M of each one of CaCl2, MgCl2 and MnCl2, and sequentially incubated for 1 h with a polyclonal anti-LMN-1 antibody (1 : 100) and a secondary antibody conjugated to peroxidase (1 : 8000), in TBST blocking buffer.
Competition with LMN-1-related peptides.
Prior to the interaction with immobilized LMN-1, parasites were pre-incubated for 30 min at 4 °C without added peptides or with 30 µg ml–1 of one of the following LMN-1-related peptides (Table 1): AG73, C16, A208 and A13 (a kind gift from Dr M. Nomizu; Nomizu et al., 1995, 1997, 1998). Alternatively, each of the peptides was added to the interaction medium. After the incubation period the wells were washed twice to remove non-attached parasites, and those which remained associated with the LMN-1-coated slides were counted by light microscopy.
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) and incubated overnight at 4 °C with 10 % trichloracetic acid (Grimbleby & Ntailianas, 1961). The resulting proteins were resuspended in dissolving buffer (16.5 mM Tris, 0.5 % SDS, 0.5 % β-mercaptoethanol, pH 6.8), quantified according to Bradford (1976), diluted in sample buffer (Laemmli, 1970), and assayed or stored at – 20 °C. SDS-PAGE analysis was performed in 7.5 % polyacrylamide mini-gels with 30 µg protein per slot. Protein bands which were separated by SDS-PAGE were electrotransferred to nitrocellulose membranes (Towbin et al., 1979). After protein blotting, the membranes were stored at 4 °C or immediately incubated with TBST blocking buffer followed by sequential incubations with LMN-1 (20 µg ml–1), a LMN-1 goat antibody (1 : 100, Santa Cruz Biotechnology), and an anti-goat antibody conjugated to peroxidase (1 : 8000, Sigma), as described above.
Epithelial cell culture.
HeLa cells were cultured in RPMI 1640 medium (Gibco Laboratories) supplemented with 25 mM HEPES and 10 % fetal bovine serum. Cells were seeded in plastic flasks, and incubated under a 5 % CO2 atmosphere at 37 °C until confluent. The resulting cell monolayers were disrupted with 0.2 % trypsin and 0.58 mM EDTA in PBS. The cells were then washed twice in culture medium plus serum and then distributed into 96-well plates (105cells per well).
Cytotoxicity assay.
HeLa cell monolayers were labelled with [3H]thymidine [4 µCi (148 kBq) per 105 cells per well] overnight, according to Singh et al. (1999). The culture medium was then aspirated, and the wells were washed twice with warm PBS prior to the interaction with T. foetus. Previously washed parasites, which were suspended in RPMI medium without serum, supplemented or not with 20 µg LMN-1 ml–1, were added to the HeLa cell monolayers (10 parasites : 1 cell). After 5 h of parasite–cell interaction at 37 °C, the interaction medium was collected, and the released [3H]thymidine was evaluated by liquid scintillation counting (Melo-Braga et al., 2003). The cytotoxicity indices were expressed as a percentage of the positive control (monolayer lysed by 0.2 % SDS in 0.2 % NaOH). The serum-free medium in which HeLa cells were maintained was collected and also used as negative control (spontaneous release). The percentage cytotoxicity was determined by the following formula: % cytotoxicity=(c.p.m.experimental/c.p.m.positive control)x100.
Statistics.
All data were analysed by using Student's t-test to identify the significance of differences between experimental and control conditions (P
0.05).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, parasite attachment further increased concomitantly with addition of LMN-1 at amounts up to 20 µg ml–1. Thus, we used 20 µg LMN-1 ml–1 to coat glass slides throughout.
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, arrows), while the usual tear-like shape was predominant in controls (Fig. 2A, C). In order to estimate the number of amoeboid parasites adhered to LMN-1 and adhered on uncoated and LMN-1-coated glass, a quantitative analysis was performed by recording the numbers of amoeboid (spread) and non-amoeboid forms of T. foetus. Contact with LMN-1 rendered more amoeboid parasites (25.3 per field) than those attached to uncoated control slides (3 per field; P<0.05, Student's t-test).
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Indirect dot-blot analyses identified the presence of LMN-1-reactive molecules in T. foetus (Fig. 3). When whole extracts of the parasite were pre-treated with 10, 50 or 100 mg ml–1 trypsin the spots corresponding to those LMN-1-binding molecules progressively disappeared (Fig. 3). By contrast, whole extracts of T. foetus pre-treated with sodium periodate did not exhibit significant alterations in the reaction (Fig. 3).
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; P<0.05, Student's t-test). The specificity of this cation-dependent cell binding was confirmed by addition of the cation chelator EDTA to the interaction medium (Fig. 4).
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Identification of LMN-1 peptides recognized by T. foetus
Parasites were incubated with or without the LMN-1-derived peptides AG73, C16, A208 or A13 (Table 1
) before the interaction with immobilized LMN-1. Alternatively, the same amount of each of these four peptides was added to the interaction medium. When the peptides were included in the interaction medium the following parasite binding rates were obtained: 49.0 %, 11.0 %, 29 % and 20.3 %, for AG73, C16, A208 and A13, respectively (Fig. 5). When the parasites were pre-incubated at 4 °C for 30 min with the peptides, and then poured into contact with LMN-1-coated slides the parasite binding rates decreased to 3.7 %, 12.3 %, 7.3 % and 9.0 %, for AG73, C16, A208 and A13, respectively (Fig. 5).
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). As a control, when the incubation step with LMN-1 was omitted, the reaction with both primary and secondary antibodies did not result in any non-specific labelling.
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; Crouch & Alderete, 1999) we investigated the possible role played by LMN-1 in the cytotoxicity exerted by T. foetus against HeLa epithelial cells. Prior to the interaction with cell monolayers, suspended parasites were incubated with or without LMN-1, and allowed to interact with [3H]thymidine-labelled HeLa cells (parasite : cell ratio of 10 : 1) Following 5 h of cell–parasite interaction we found that the cytotoxicity exerted by T. foetus was significantly increased when LMN-1 was present in the interaction medium. Monolayers formed by HeLa cells showed a cytotoxicity rate of 22.9±0.7 % when LMN-1-treated T. foetus was assayed, compared to a cytotoxicity rate of 15.9±0.4 % with untreated parasites. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; López et al., 2000). T. foetus, as well as other cavity parasites, appears to attach tightly to host tissue to overcome removal by physical forces. Therefore, the adhesion of T. foetus to both epithelial cells and ECM is an essential step for parasite maintenance in the host. The major component of basal membrane, LMN-1, may play a role during T. foetus binding and invasion of host tissue. As previously described (Silva-Filho et al., 1988), we observed that after a few minutes of interaction between T. foetus and immobilized LMN-1, most of the parasites were physically associated with the glycoprotein (data not shown), and the number of parasites attached to LMN-1 was directly related to the time spent by the parasite to spread onto the LMN-1-coated surfaces. Additionally, a correlation between the number of parasites attached to LMN-1 and the amount of LMN-1 immobilized onto slides was observed (Fig. 1). The highest T. foetus adhesion index was obtained when 20 µg LMN-1 ml–1 was used to coat the slides. Such results led us to consider that T. foetus attachment on LMN-1 is a specific process involving ligand–receptor binding.
A morphological alteration in T. foetus was observed when it was attached to immobilized LMN-1 (Fig. 2). The occurrence of amoeboid forms among parasites associated with LMN-1-coated slides strongly indicates that the association of T. foetus with LMN-1 results in an adhesive process, in which parasites are led to spread onto the immobilized glycoprotein. This seems to lead to a remodelling of both the parasite surface and also the meshwork formed by LMN-1. Trichomonas vaginalis also alters its morphology as soon as it adheres to host cells or some ECM glycoproteins (Schwebke & Burgess, 2004; Crouch & Alderete, 1999; Petrin et al., 1998). Despite the potential importance of membrane trafficking and intracellular signalling, little is known about the molecular mechanisms underlying such morphological alteration phenomena (Arroyo et al., 1993; Lal et al., 2006).
Different types of LMN-1-binding molecules have been found in micro-organisms and mammalian cells, including LMN-binding proteins (Mercurio, 1995), integrins (Hynes, 2002; Rao et al., 1992; Hernandez-Ramirez et al., 2000) and receptins (Kronvall & Jonsson, 1999). In the present study we were able to identify the chemical nature of T. foetus LMN-binding molecules, and their dependence on divalent cations. The cation dependence of T. foetus–LMN-1 binding suggests the presence of integrin-like molecules in T. foetus, as already described in Entamoeba histolytica (Hernandez-Ramirez et al., 2000). The occurrence of integrin-like molecules in T. foetus is currently under investigation in our laboratory.
The presence of different sites in the LMN-1 polypeptide chain as possible binding sites for prokaryotic and eukaryotic cells has been reported (Nomizu et al., 1995, 1997, 1998). Futhermore, oligosaccharides associated with LMN-1 (Fujiwara et al., 1988) have been also considered as important LMN-1 recognition sites by lectin-like molecules (Elola et al., 2005; Ferreira et al., 2006). Some lectin-like molecules recognize β-galactose and lactose residues of LMN-1 (Elola et al., 2005; Woo et al., 1990; Gu et al., 1994); therefore the possibility that T. foetus may recognize LMN-1 through oligosaccharide chains on ECM molecule was explored. The presence of β-lactose or galactose in the interaction medium did not alter the binding of the parasite to LMN-1, excluding the possibility that T. foetus recognizes LMN-1 through these oligosaccharide moieties. On the other hand, the presence in the interaction medium of oligopeptides corresponding to amino acid sequences in the 
1 and 
1 chains of LMN-1 partially inhibited the binding of the parasite to the immobilized protein (Fig. 5, competition assay). A high binding inhibition was also observed when T. foetus was pre-incubated with each of these peptides (Fig. 5, pre-incubation assay). These data indicate the possibility that T. foetus may recognize LMN-1 through different well-known amino acid binding sequences of the molecule, as previously observed for other eukaryotic cells (Mercurio, 1995; Nomizu et al., 1995, 1997, 1998; Ferreira et al., 2006).
Five LMN-1-binding proteins, of approximately 220, 200, 130, 125 and 80 kDa, were detected in parasite homogenates. The number of LMN-1-binding proteins in T. foetus might be specifically related to the number of different cell recognition sites on the LMN-1 molecule, but the occurrence of a promiscuous surface glycoprotein of T. foetus involved in the recognition of LMN-1 cannot be ruled out. It remains unknown whether the chemical entity present in T. foetus, which we have named LMN-binding protein(s), corresponds to a multiligand recognition complex.
We demonstrated that the cytotoxicity of T. foetus to HeLa cells increased when the parasite interacted with LMN-1. The increase in cytotoxic effect might be related to an enhancement in cytoadhesion, since LMN-1-treated T. foetus adhered more to mammalian cells than did untreated ones (Silva-Filho et al., 1988). However, it appears that the cytotoxicity exerted by T. foetus is not only a cytoadhesion-dependent phenomenon (Silva-Filho et al., 1989). The binding of T. foetus to LMN-1 might trigger a signalling cue that could culminate in the upregulation of cytotoxic factors, such as the proteases released during interaction of trichomonads with mammalian cells (Alvarez-Sanchez et al., 2000).
In summary, our studies identified five LMN-1-binding proteins in T. foetus that recognize LMN-1 by at least four different binding sequences. Taken together, these findings suggest an organized interaction complex in our parasitic protozoa model that is similar to those found in mammalian cells. T. foetus adhesion on LMN-1 biofilm was characterized as a cation-dependent phenomenon, by changes in T. foetus morphology and cytotoxicity, suggesting that the parasite–ECM interaction leads to an exchange of signals from ECM to the parasite. Moreover, in vivo LMN-1 could serve as a molecular track to T. foetus, probably guiding it to anatomic sites outside the urogenital tract.
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ACKNOWLEDGEMENTS |
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Edited by: J. Tachezy
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Received 14 December 2007;
revised 8 February 2008;
accepted 7 April 2008.
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